Darwinian evolution generates diversity and allows improvement of an individual's component parts in a changing environment. One approach to designing enzymes with altered specificities is to utilize evolution in a context that can be managed in vitro. This kind of approach requires a linkage between genotype and phenotype and a strategy for selection and enrichment of distinct genotypes. Methods based on these considerations, include phage display (see for example Smith, Science 228:1315-1317 (1985), and U.S. Pat. No. 7,211,564); mRNA display (Hanes et al., Proc. National Academy Science 94:4937-4942 (1997), and Tawfik et al., Nat Biotechnol. 16:652-656 (1998)); and ribosomal display (Roberts et al. Proc Natl Acad Sci USA 94:12297-12302 (1997)). These methods utilize display of a desired phenotype on the surface of a particle that is capable of replication.
An alternative method of directed evolution that has been referred to as in vitro compartmentalization (IVC) relies on forming an emulsion of aqueous droplets in oil where the aqueous droplets contain a controlled amount and type of polynucleotide together with, at a minimum, a transcription and translation system to permit expression of any genes encoded by the encapsulated polynucleotides. The water dropleits in these emulsions are also referred to as microcapsules in at least some of the publications listed below. The protein product of transcription and translation can then be detected by some form of assay that preferably permits enrichment of the target gene which may be initially present at an amount as low as 1 in 1010 droplets (see for example, Doi et al. Nucleic acids Res 32:e95 (2004), U.S. Pat. Nos. 6,184,012, 6,489,103, 6,495,673, 7,138,233, and 7,252,943, U.S. Publication Nos. 2005/0221339, 2006/0153924, 2006/0154298, 2006/0078893, 2007/0077,572, 2007/0092914, 2007/0184489, and 2008/0004436, and International Application Nos. WO 2006/040551 and WO 2006/051552).
If the problem of selection and enrichment can be solved, the ability to stably create as many as 109˜1010 individual aqueous droplets in a single emulsion where each droplet contains a different molecule from a library of molecules could provide a rapid screening method for target genes encoding a desired phenotype in an extensive sequence space.
Unfortunately, reported screening and enrichment methods using IVC have been disappointing. For example, Doi et al. described a screening method for selecting and enriching for a genotype expressing a restriction endonuclease phenotype. Doi et al. used a DNA polymerase to incorporate dUTP-biotin to the sticky ends generated by restriction endonuclease cleavage permitting strepavidin affinity purification. Only 10-fold enrichment of a single polynucleotide was achieved in one round of in vitro compartmentalization. Consequently, a large number of rounds of enrichment were required to select an active phenotype, FokI, from a randomized FokI library.
In vitro compartmentalization using emulsions offers a potentially powerful method of directed evolution if selection of a protein activity and its enrichment in a solution can be optimized to allow efficient recovery of a target gene from the extensive sequence space.